Modifications were made in agreement with US EPA to address validity of test material and in testing procedures

Principles of method if other than guideline:

A previous algal test showed decrease in measured test material to levels below detection levels before test termination at 96 hours. This test was designed to provide better estimation of concentrations over time. Modifications were discussed with US EPA.

The test protocol conformed to US EPA TSCA Test Guideline 797.1050, 1985 as amended 20 May 1987 with the following exceptions: - Modifications to address validity of test material - Test solution volumes were 100 ml in 125 ml flasks to minimize head space - Teflon stoppers were used to prevent gas exchange - Additional exposure at the termination of the study to examine algistatic/cidal action was excluded as meaningful exposure concentrations were not present - Test sampling was conducted daily, rather than just at beginning and end to better assess exposure concentrations. Study results were presented in terms of initial concentrations and as time weighted averages

Additional Modifications: - Stock cultures were maintained, as well as test cultures, on a rotary shaker rather than just shaken twice daily. - At request of US EPA. Na2EDTA.2H2O was added to the test medium at a concentration of 0.38 mg/L to promote algal growth. - The strain for the test species Selenastrum capricornutum was not available

GLP compliance:

yes (incl. certificate)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: at test initiation, samples were collected from the volumetric flasks used to prepare the test solutions. At each 24-hour interval during the test, three replicates from each test concentration, controls and solvent controls were selected impartially. Aliquots of approximately 50 ml were removed from each of the three replicates and composited for analysis. One of the nine 1.0 mg/L flasks (without algae) was sampled at test initiation. At 2,4,8,12,24,48, 72 and 96 hours, one remaining flask was selected impartially and analyzed for DTBP to determine loss from solution in the absence of algae. At each sampling interval, three Quality Control Samples were prepared by adding known volumes of stock solution to fresh algal growth medium. The QC samples remained with the test samples throughout analysis.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION - Method: A radiolabelled stock solution was prepared by transfer of the entire amount of labelled DTBP in one vial (1.159 g) into a 25 mL volumetric flask, and diluting to volume with acetone. Triplicate 10 uL aliquots of this stock solution were analyzed by liquid scintillation counting. The measured mean stock concentration was 41.9 mg/mL. The radiochemical purity of the stock solution was 97.4% using HPLC with radiometric detection. The stock solution was kept at about -20 degrees C until use.A non radio-labelled stock solution of 158 mg/mL was prepared by weight 1.5824 g DTBP into a 10 mL flask and diluting to volume with acetone. A second non-labelled stock solution of 58.1 mg/mL was prepared by weighing 5.818 g DTBP into a 10 mL flask and diluting with acetone.

- Test solutions: The highest test concentration (10 mg/mL) was prepared by combining 0.10 mL of the 41.9 mg/mL radiolabelled stock with 0.10 mL of the 158 mg/mL non-labeled stock solution and diluting to 2000 mL with sterile MBL media. The second highest test concentration (5 mg/mL) was prepared by combining 0.10 ml of the 41.9 mg/mL radiolabelled stock solution with 0.10 mL of the 58.1 mg/mL non-labelled stock solution and diluting to volume with the sterile MBL medium. The remaining test concentrations were prepared by combining the appropriate volume of the 41.9 mg/mL radiolabelled stock solution with an appropriate volume of acetone and diluting to 2000 ml with MBL media.

- Eluate: acetone, MBL media- Controls: Algal growth medium alone for control; solvent control contained 0.10 mL/L acetone. A 1 mg/L solution was prepared by diluting 0.024 mL of the 41.9 mg/ml radiolabelled stock to 1000 MBL medium - this solution was used to measure concentration of test material over time in absence of algae.- Chemical name of vehicle: acetone- Concentration of vehicle in test medium: 0.10 ml/L acetone

TEST SYSTEM - Test vessel: Special exposure systems to minimize loss of test article - Type: closed - Material, size, headspace, fill volume: 125 ml Erlenmeyer glass flasks with teflon coated stoppers containing 100 ml of test solution. The stoppers did not allow gas exchange, and the additional volume of test solution minimized head space. A set of three flasks were prepared as reference cultures in standard test vessels with stainless steel stoppers and containing only 50 ml of control solution. - Aeration: no

- Initial cells density: 1 x 10^4 cells/ml in test solutions - Control end cells density: same as in test solutions - No. of vessels per concentration (replicates): 12 replicates per concentration - No. of vessels per control (replicates): 12 per control - No. of vessels per vehicle control (replicates): 12 per solvent control

TEST CONCENTRATIONS- Test concentrations definitive test based on results from an earlier definitive test conducted at the testing laboratory with the same species (Report no: 88-11-2846)

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (0.46 to 3.5)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (1.0 to 12)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL 1.5 to 9.3)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (1.9 to 6.1)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (0.27 to 2.5)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (0.59 to 4.8)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (0.58 to 3.6)

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CL (0.63 to 1.8)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.64 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Details on results:

Algal Growth: - Control cultures and solvent control cultures used the same closed systems as did the test cultures. At 96 hours, control cultures averaged 19 x 10^4 cells/mL, solvent control cultures averaged 21 x 10^4 cells/mL as compared to reference cultures (in normal topped tubes, with normal surface areas) which averaged 83 x 10^4 cells/mL. The lower densities in the controls and solvent controls reflect carbon dioxide limitation in sealed flasks. - Cell densities in test cultures ranged from 2 to 20 x 10^4 cells/mL at 96 hours, and generally followed the concentration gradient. Cell growth was not completely inhibited in any of the test concentrations.

Cell Abnormalities: - Small cells and clumping were observed in algal cultures exposed to 0.33 to 1.2 mg/L and in the control and solvent control. -Cell fragments were observed in cultures exposed to 2.1 and 2.9 mg/L (initial measurements) -Bloated and uncurled cells were observed at the highest test concentration (7.2 mg/L) - Reference cells were normal in size and shape and appeared healthy

Reported statistics and error estimates:

- First stage: Control cell densities were compared with solvent control cell densities at each sampling interval using Student's t-test. If the densities did not differ, data from solvent controls were used for further analysis.- Second stage: EC50 values based on initial measured concentrations and on time-weighted-averages were calculated from cell densities at 24, 48, 72 and 96 hours of exposure. For each period, the EC10, EC50, and EC90 values and their 95% confidence limits were determined by linear regression of response (percent reduction in cell density as compared with controls) versus the initial or TWA concentration over the range of test concentrations where a clear exposure-response relationship was observed. Four linear regressions were estimated based on a) untransformed data b) untransformed response versus logarithm-transformed concentration c) probit-transformed response versus untransformed concentration and d) probit-transformed response versus logarithm-transformed concentration. The regression that best fit the data was selected based on highest coefficient of determination. This regression equation was then applied to estimate the EC values and their 95% confidence limits using the method of inverse prediction. - The No Observed Effect Concentration based on initial measured concentrations and on time weighted average concentrations was determined by using one-way analysis of variance and Dunnett's test if all groups had equal numbers of replicates (i.e. if control data were not pooled) or using Bonferroni's test if groups had unequal numbers of replicates (control data were pooled). Before conducting Analysis of Variance, data were checked for normality using the Chi-Square test and for homogeniety of variance using the Hartley test. If assumptions of normality and homogeneity of variance were not met, the NOEC was determined by using the nonparametric Kruskal-Wallis test.

Concentrations of 2,6 DTBP measured by HPLC with UV detection during the 96 hour toxicity test

with Selenastrum capricornutum

Nom. Conc. (mg/L)

Measured Concentration (mg/L)

0-hour

24 hour

24-hour TWA

48 hour

48 hour TWA

10

7.2

2.2

4.7

0.83

3.4

5.0

2.9

1.3

2.1

0.32

1.5

2.5

2.1

0.81

1.5

0.20

1.0

1.3

1.2

0.40

0.81

0.059

0.56

0.63

0.63

0.19

0.41

0.059

0.30

0.31

0.33

< 0.12

0.21

<0.050

0.14

Solvent Control

< 0.14

< 0.12

-

<0.050

-

Control

< 0.14

< 0.12

-

<0.050

-

1.0 mg/L

0.93

0.15

-

<0.050

-

a Time weighted average (TWA) concentration was calculated by averaging the measurement for the stated interval with the measurement from the previous interval

b Measured concentration at this interval was below the detection limit and 0 was entered in the TWA equation

A previous acute algal toxicity study conducted at this laboratory (SLS report number 88 -11 -2846) showed dose response effects in Selenastrum capricornutum exposed to 2,6 -di-tert-butylphenol (DTBP). However, loss of test material over the course of the 96 hour study caused measured concentrations to fall below limits of detection such that establishment of actual exposure concentrations could not be done. This study was designed to provide a better estimate of exposure concentrations over time so that time-weighted average concentrations could be calculated and used in determination of EC50 values. The study design was modified to prevent volatilization of the test article from solutions by using sealed culture flasks with limited head space. Because the lack of gas exchange was expected to impair the growth of algae in the sealed system, three reference control flasks (with standard closures permitting gaseous exchange) were used to confirm test culture viability. An additional series of flasks without algal inoculum permitted monitoring of DTBP concentrations over time in the absence of algal action. Analytical methods used were HPLC with UV detection, HPLC with radiometric detection, and liquid scintillation counting. A time weighted average concentration was calculated for each set of test solutions at each sampling interval. Initial measured concentrations based on HPLC-UV analysis were 7.2, 2.9, 2.1, 1.2, 0.63, and 0.33 mg/L for nominal concentrations of 10, 5, 2.5, 1.3, 0.63, and 0.31 mg/L. At test termination only the two highest concentrations contained measurable amounts of test material (detection limit 0.025 mg/L). The 96 hour time weighted averages were 2.2, 0.95, 0.64, 0.34, 0.18, and 0.086 mg/L. Test vessels containing 1.0 mg/L but without algae were tested at 0,2,4,8,12,24, 48, 72 and 96 hours to determine loss of DTBP in the absence of algae. After 4 hours, measured concentrations in those solutions declined somewhat faster than those in test solutions with algae. The decline in DTBP values in the test solutions thus were not caused by the algae in those solutions. Supplemental analyses of test solutions were performed using HPLC-RAM to confirm the HPLC-UV results and to identify any radiolabeled degradates. Although measured concentrations decreased over time, no radiolabeled degradates were detected. It was inferred that the degradates of DTBP were volatile and released to the headspace. Because all the 14C in the test solutions occurred as DTBP, total 14C measurements were employed for the analyses of the 48, 72, and 96 hour samples to maximize the analytical sensitivity. The disappearance half-lives were calculated from the slope of the log concentrations versus time regressions. Half-lives ranged from 14 to 17 hours. The half-life in the 1.0 mg/L uninoculated solutions was estimated to be 8.4 hours. Cell counts in control and solvent control cultures ranged from 19 to 21 x 10^4 cells/mL. Cell densities in the reference cultures (standard topped flasks and normal surface area) averaged 83 x 104 cells/mL at 96 hours, and cells appeared normal in size and shape. The reference culture densities were typical of standard condition tests. The lower densities in control and reference controls reflected carbon dioxide limitation in the sealed flasks. Cell densities in the test cultures ranged from 2 to 20 x 10^4 cells/mL at 96 hours and generally followed the concentration gradient. Cell growth was not completely inhibited at any concentration, and cells in the test concentrations (up to 1.2 mg/L) and in control and solvent control cultures appeared small and some clumping was noted. Some cell fragments were observed in the 2.1 and 2.9 mg/L and at the highest treatment level (7.2 mg/L) some cells were bloated and uncurled. Based on initial measured concentration, the 96 hour EC50 was calculated to be 3.9 mg/L (95% confidence limit 1.9 to 6.1 mg/L). The 96 hour NOEC was calculated to be 2.1 mg/L. Based on time weighted average concentrations, the 96 hour EC50 was 1.2 mg/L (95% confidence interval 0.63 to 1.8 mg/L) and the 96 hour NOEC was 0.64 mg/L.

Description of key information

96 h EC50: 1.2 mg a.i./L

96h-NOEC = 0.64 mg a.i./L

2,6-DTBP is algistatic rather than algicidal.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.2 mg/L

EC10 or NOEC for freshwater algae:

0.64 mg/L

Additional information

Two studies were conducted using the guideline standard test organism, Selenastrum capricornutum, over 96 h.

The study conducted by Giddings J.M. (1989) providing 96h-EC10 and EC50 values of 0.18 and 0.56 mg a.i./L, respectively. This study had poor recovery of test substance during analytical dose verification analysis. The reliability of the study was therefore reduced. Therefore this study was considered to be supporting only.

The selection of the key study for aquatic algae and cyanobacteria has no impact on the PNEC calculation and the risk assessment since the both the lowest EC50 and NOEE/EC10 from the algal studies was higher than the EC50 and NOEC for invertebrates. Therefore, the outcome of the algae studies was not relevant for the risk assessment of 2,6 -DTBP. 2,6-DTBP is algistatic rather than algicidal.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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